摘要
本文继大肠杆菌表达含凝血酶切点的人重组IL-3融合蛋白成功的基础上进一步探讨了天然型rhIL-3的纯化。超声破碎细菌细胞得包涵体,经洗涤处理可使包涵体纯度达90％,用8mol/L尿素变性溶解包涵体沉淀后直接稀释复性,再超滤浓缩、凝血酶消化,释放天然型rhIL-3。经DEAE Sepharose Fast Flow和Sepharcyl-100 HR层析得到天然型IL-3,纯度达96％,回收率20％以上,具有刺激正常人骨髓细胞形成集落的活性。本实验为大批量重组IL-3的生产创造了条件。
A protocol for purifying rhIL-3 from E.coli was developed following the success of expressing rhIL-3 containing thrombin-recognition site immediately upstream of IL-3. Inclusion bodies (IB)collected after the breakage of bacteria through sonication were subjected to repeated washing,and reached a purity of approx 90%. IB solubilized in the presence of 8mol/L urea were diluted with renaturation solutions so that folding process initiated. Concentrated IL-3 were digested with thrombin and authentic rhIL-3 released. By means of DEAE Sepharose Fast Flow and Sephacryl-100 HR, authentic IL-3 were obtained with a purity of over 95%. The total recovery of this protocol is approx 20&. The purified products were highly effective in stimulating the colony formation of human bone marrow cells. This study also provided useful parameters for massproduetion of rhlL-3.
基金
863计划资助课题
关键词
白细胞介素3
提纯
大肠杆菌
Recombinant human IL-3
Thrombin
Purification of protein
E. coli
